1. Field of the Invention
The present invention relates to a method for determining an increased likelihood of the presence of chronic fatigue syndrome (CFS) in an individual by determining the presence of elevated levels of Mycoplasma DNA in peripheral blood mononuclear cells.
2. Description of the Related Art
Chronic Fatigue Syndrome (CFS) is an illness with increasingly reported frequency in the United States and other industrialized countries (Straus, Rev. Infect. Dis. 13(Suppl. 1):S2-S7, 1991). CFS is characterized by prolonged and debilitating fatigue with multiple non-specific symptoms such as headaches, recurring sore throats, muscle and joint pains and cognitive complaints. Profound fatigue, the hallmark of the disorder, can appear suddenly or gradually and persists throughout the course of the illness. Unlike the short-term disability of an acute viral infection, for example, CFS symptoms by definition linger for at least six months and often for years (Fukuda et al., Ann. Intern. Med. 121:953-959, 1994). Physicians can evaluate patients with persistent fatigue of undetermined cause using guidelines developed by the international CFS study group (Fukuda et al., Fed. Pract. 12:12-17, 1995).
It has been well documented that individuals who suffer from fibromyalgia (FMS) exhibit many of the same symptoms found in atypical CFS (Buchwald et al., Arch. Intern. Med. 154:2049-2053, 1994; Ziem et al., Arch. Intern. Med. 154:1913, 1995) in which a patient has 6 or 7 tender points. These two illnesses are so similar that for years many medical practitioners have considered them to be the same condition.
Despite multidisciplinary investigations of CFS, its etiology remains unknown and no specific diagnostic tests or therapies for CFS exist. In about one third of cases, the sudden onset follows a respiratory, gastrointestinal, or other acute infection with flu-like symptoms, including mononucleosis (Mawle et al., Infect. Agents Dis. 2:333-341, 1994). No published data implicate a specific virus or other microbes as the cause of CFS. However, it appears that infectious agents, among other stressors, can precipitate the syndrome (National Institutes of Health Publication No. 96-484, 1996). A variety of common viruses can be reactivated in some CFS patients, including HTLV-II, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV) 1 and 2, and human herpes viruses 6, 7 and 8. It is believed that virus reactivation could be occurring secondarily to some immunologic disturbance (National Institutes of Health Publication No. 96-484, 1996; Nicolson et al., Int. J. Occup. Med. Immunol. Toxicol. 5:69-78, 1996).
Mycoplasmas are bacteria which belong to the class Mollicutes. They are the smallest free-living, self-replicating bacteria known. They have no cell wall and a very limited genome of between 600 and 1,500 kilobases which makes them highly dependent on their host for survival. The mycoplasma species M. fermentans, M. hominis and M. penetrans have been isolated from individuals suffering from primary atypical pneumonia, urogenital infections, rheumatoid arthritis (RA) and AIDS-related infections (Hayes et al., Infect. Immun. 64:3419-3424, 1996; Schaeverbeke et al., Br. J. Rheumatol. 36:310-314, 1997; Montagnier et al., Clin. Infect. Dis. 17(Suppl. 1):S309-315, 1993).
There is an ongoing need for methods of detecting CFS infection. The present invention addresses this need.
One embodiment of the present invention is a method for determining an increased likelihood of the presence of chronic fatigue syndrome (CFS) and/or fibromyalgia (FMS) in an individual, comprising the steps of: isolating peripheral blood mononuclear cells (PBMC) from said individual; and determining an amount of a Mycoplasma species present in the PBMC from the individual compared to an amount of Mycoplasma species present in PBMC from a control individual known not to have CFS or FMS, wherein an increase in the amount in the individual compared to the control individual indicates an increased likelihood of the presence of CFS and/or FMS. Preferably, the Mycoplasma species is Mycoplasma fermentans. In one aspect of this preferred embodiment, the determining step comprises detection of Mycoplasma polynucleotides. Preferably, the determining step comprises use of polynucleotide amplification. In another aspect of this preferred embodiment, the determining step comprises amplification of a DNA fragment specific to a plurality of Mycoplasma species. Preferably, the determining step comprises amplification of a 206 base pair fragment specific to Mycoplasma fermentans. In one aspect of this preferred embodiment, the amount of the Mycoplasma species present in PBMC from a control individual known not to have CFS is a mean average of a plurality of control individuals. Preferably, the increase is at least about 25%. More preferably, the increase is at least about two-fold. Most preferably, the increase is at least about three-fold. In one aspect of this preferred embodiment, the amount of Mycoplasma species is calculated as a genome copy number. Preferably, the determining step comprises quantitative competitive PCR. Alternatively, the determining step comprises Southern blotting. Advantageously, the quantitative competitive PCR comprises use of the oligonucleotide primers having sequences shown in SEQ ID NOS: 9 and 10.
The present invention also provides a method for determining an increased likelihood of the presence of chronic fatigue syndrome (CFS) in an individual, comprising the steps of: isolating blood serum from said individual; and determining the amount of anti-Mycoplasma antibodies in blood serum, wherein an increase in the amount compared to a control individual known not to have CFS indicates an increased likelihood of the presence of CFS. Preferably, the determining step comprises enzyme- linked immunosorbent assay. In one aspect of this preferred embodiment, the antibodies are anti-M. fermentans antibodies. Advantageously. the anti-M. fermentans antibodies recognize M. fermentans P29 surface lipoprotein. In another aspect of this preferred embodiment, the amount of antibodies specific to Mycoplasma present in serum from a control individual known not to have CFS is a mean average of a plurality of control individuals. Preferably, the increase is at least about 25%. More preferably, the increase is at least about two-fold. Most preferably, the increase is at least about three-fold.